Mice ablated for nonmuscle myosin II-B (NM II-B) died as embryos by E14.5 due to congenital heart defects. NM II-B knockout hearts showed a hypoplastic myocardium with a marked reduction in cardiac myocyte proliferation rate which we had previously attributed to a failure of cardiac myocyte cytokinesis. Interestingly, replacement of wild-type NM II-B with the motor-impaired R709C NM II-B in mice rescued cardiac myocyte cytokinesis but not the hypoplastic myocardium. The epicardium plays a critical role in myocardial proliferation. In addition to expression in cardiac myocytes, NM II-B is also detected in the epicardium. We thus examined epicardium formation and function in R709C NM II-B hearts and NM II-B knockout hearts. Both the mutant and knockout epicardium showed a focal detachment from myocardium associated with reduced amounts of epicardial E-cadherin and fibronectin. Importantly the expression of epicardial FGF-9 was markedly diminished in NM II-B mutant and knockout epicardium. Thus defects in epicardium formation impair epicardial FGF-9 signaling consequently limiting cardiac myocyte proliferation and contributing to the development of a hypoplastic myocardium. NM II-B is enriched at the cell-cell junctions between epicardial cells. Super-resolution structured illumination microscopy (SIM) analysis shows a paired NM II-B alignment between epicardial cells, suggesting a role for NM II-B in regulating epicardial cell-cell adhesion. Ablation of NM II-B results in a loss of E-cadherin at epicardial cell-cell adherens junctions and disorganization of tight junctions. NM II-B also plays important roles in normal epicardial epithelial-mesenchymal transition (EMT). In in vitro epicardial explants, ablation of NM II-B impairs epicardial-derived cell migration into collagen gels following fetal bovine serum stimulation. Interestingly NM II-B knockout explants show a decreased expression of mesenchymal genes such as Cdh6 (K-cadherin), Acta2 (smooth muscle &#61537;-actin, SMA), Tagln (SM22&#61537;), and Postn (periostin), although no changes in epithelial genes and major EMT master genes were found compared to wild type explants. Therefore NM II-B is directly involved in regulation of epicardial EMT in addition to its role in cell migration. Defects in epicardial EMT in developing mouse hearts result in abnormalities in coronary vessel formation in NM II-B ablated mice. The importance of epicardial expression of NM II-B in coronary vessel formation was further confirmed in mice conditionally ablated for NM II-B in epicardial cells using WT-1-cre mice. Although the hearts of the conditional knockout mice show no evidence of cardiac myocyte cytokinesis defects indicating an intact NM II-B function in cardiac myocytes, they still develop defects in coronary vessel formation. Our results demonstrate the essential roles of NM II-B in the epicardium during heart development.